Fig 1: PU.1 (SPI1)-mediated formation of a pro-fibrotic CAF stroma is associated with CRPC progression.A Bar plots representing SPI1 gene expression across different PCa stages based on a cross-study comparison of multiple bulk RNA-seq datasets performed through the Prostate Cancer Atlas 61. The logarithm of counts per million reads (log CPM) is reported on the y-axis. Mean, standard deviation, and every single measurement (dots) are reported. Ordinary one-way ANOVA with Tukey’s multiple comparisons, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). ARPC: AR-positive castration-resistant prostate cancer, DNPC: double-negative prostate cancer, NEPC: neuroendocrine prostate cancer. B Bar plots showing human SPI1 normalized gene expression level across prostate cell types (epithelial, B cells, T-cells, mast cells, myeloid cells, CAFs, and endothelial cells) in all PCa stages. Mean, standard deviation, and every single measurement reported. C Bar plots showing human SPI1 normalized gene expression level across the indicated stromal cell types, comparing its expression in primary PCa, CRPC, and NEPC samples. For visualization purposes, the mean and standard error of the mean with no individual values were plotted. Bar plots with standard deviation and individual values are in Supplementary Fig. 11D. Two-way ANOVA with Šídák’s multiple comparisons test, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). D Boxplots showing PTPRC, ACTA2, S100A4, and COL1A1 (columns) normalized expression levels across the indicated cell types (rows) in CRPC samples. Annotated single cells have been stratified for their SPI1 status; off: no SPI1 expression detected, on: SPI1 expression detected. B–D Data have been obtained from the single-cell RNA sequencing Prostate Cancer Cell Atlas (PCCAT)62. apCAF: antigen-presenting CAF, dCAF: differentiated CAF, iCAF: inflammatory-like CAF, mCAF: matrix-associated CAF, myoCAF: myofibroblast-like CAF, rCAF: reticular CAF, SMC: smooth-muscle cells. These subpopulations were defined in ref. 62. E Graphical summary of the proposed cellular and molecular stromal alterations in CRPC progression. i CRPC-associated stroma shows a pro-fibrotic phenotype compared to androgen-sensitive PCa-associated stroma. ii CRPC tumor cells induce the formation of a pro-fibrotic stroma enriched with immune cells, iCAF, and myCAF, which produces inflammatory cytokines, and ECM proteins (e.g., collagen-I, TNC). This reactive stroma might, in turn, promote tumor cell motility and invasive potential (intra-tumoral infiltration). iii TGFβ-signaling and the transcription factor PU.1 are involved in the acquisition of the reactive stroma phenotype (activated iCAFs and myCAFs), which supports CRPC tumor cell growth and motility (indicated by the black dashed arrow). Pharmacological inhibition of PU.1 transcriptional activity (DB1976 compound) prevents the reactive stromal phenotype induction (full red arrow), thereby indirectly impacting tumor cell growth and motility (dashed red arrow). Legend of the described cellular and non-cellular components is reported at the bottom. Created in BioRender. Karkampouna, S. (2026) https://BioRender.com/vcjuqbx.
Fig 2: LAPC9-derived CAFs support PNPCa tumor cell viability in hybrid tumor-CAF 3D collagen co-cultures.A, B In the experiments described, PNPCa and LAPC9 organoids were pre-formed in ULA and subsequently transferred into a collagen matrix upon organoid formation (after 5–7 days). A. Immunofluorescent staining of FFPE sections of PNPCa and LAPC9 tumor-CAF co-cultures after 7 days of collagen co-culture. Left panels: collagen type-I (blue channel, represented in white), cyclophilin A (green), HLA (red). Middle panels: E-cadherin (blue channel, represented in white), Ki67 (green), α-SMA (red). Right panels: AR (blue channel, represented in white), α-SMA (green), TNC (red). TOPRO-3 (far red channel, represented in blue) marks the nuclei. Scale bar: 100 µm. B Heatmap representing the RNA expression of Acta2, Col1a1, Col2a1, Col5a1, Mmp3, and Mmp9 in PNPCa and LAPC9 stromal cells after PDX tissue digestion (p0), and in collagen tumor-CAF 3D co-cultures (3D). The color code reports the mean of the logarithm of the relative expression (log(2−∆Ct)) of the indicated gene to Actb (housekeeping gene), with blue indicating lower expression, and red higher expression. Row-wise Z-score normalization per gene across samples was performed for the heatmap visualization, n = 2 biological replicates with each n = 3 technical replicates per condition. C–J In the experiment described, hybrid co-cultures were first generated in ULA and subsequently transferred into a collagen matrix upon organoid formation (Day 5–7). C Representative brightfield and red fluorescence images of PNPCa tumor + LAPC9 CAF and PNPCa + matched CAF collagen co-cultures. PNPCa tumor cells have been labeled with BioTracker™ 555 Orange Cytoplasmic Membrane Dye prior to recombination with PNPCa or LAPC9 stromal cells. Scale bar: 50 µm. D Measurement of PNPCa organoid average area size (µm2) in PNPCa tumor (human; hu) cell monocultures, PNPCa tumor + PNPCa CAF (mouse; ms), and PNPCa tumor + LAPC9 CAF co-cultures performed with Fiji software on multiple brightfield images of n = 2 biological replicates. Mean, standard deviation, and every single measurement (dots, technical and biological replicates) are reported. Ordinary one-way ANOVA with Tukey’s multiple comparison, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). E–G RT-qPCR was performed on RNA extracted from PNPCa tumor (hu) + PNPCa CAF (ms), and PNPCa tumor + LAPC9 CAF collagen co-cultures, PNPCa CAF, and LAPC9 CAF collagen mono-cultures using mouse-specific primers. Mean, standard deviation, and every single value (technical and biological replicates) of the logarithm of the relative expression (log(2−∆Ct)) of the indicated genes relative to Actb (housekeeping gene) are reported, n = 5 biological replicates with each n = 3 technical replicates for co-culture conditions, n = 2 biological replicates with each n = 3 technical replicates for mono-culture conditions. Ordinary one-way ANOVA with Tukey’s multiple comparison, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). H Representative flow cytometry analysis (dot-plots) of PNPCa + LAPC9 CAFs, PNPCa + matched CAFs, or PNPCa monocultures, performed at co-culture Day 4. Annexin V-FITC staining was used to discriminate live (Annexin V -) and apoptotic (Annexin V +) tumor cells (BioTracker555-positive) and CAFs (BioTracker555-negative). I-J Fold change of the fraction of viable (I) and apoptotic (J) cells compared to the monoculture control for n = 3 independent flow cytometry experiments (biological replicates). Mean, standard deviation, and every single measurement (dots) are reported. Statistics: Friedman test with Dunn’s multiple comparison.
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